The Zhu laboratory has established a novel experimental system using an RD29A:LUC transgene in the model organism Arabidopsis thaliana to study transcriptional gene silencing. Three repressor of silencing mutants (ros1, 2 and 3) where the normally active RD29A:LUC transgene and the endogenous RD29A gene undergo transcriptional gene silencing have been isolated. ROS1 has already been identified and shown to encode a DNA demethylase that works by a base-excision mechanism. Here I propose to characterize the ros2 and ros3 mutants with regard to DNA methylation, histone modification and production of small RNAs from the RD29A promoter. The ROS2 and ROS3 genes will be identified by map based cloning. The functions of the gene products and their mechanism of action in gene silencing will be characterized. We will also use genetic approaches to identify loci that genetically interact with ROS1. First, double mutants of ros1 and known mutants that affect gene silencing, DNA methylation or chromatin structure will be constructed and the expression level of RD29A:LUC analyzed. Second, newly isolated mutants that suppress the gene silencing phenotype of ros1 will be characterized using similar methods as applied to ros2 and ros3.